Porphyria Research Articles

Novel frameshift variant (c.409dupG) in SLC25A38 is a common cause of congenital sideroblastic anaemia in the Indian subcontinent

AimsCongenital sideroblastic anaemias (CSAs) are a group of rare disorders with the presence of ring sideroblasts in the bone marrow. Pathogenic variants are inherited in an autosomal recessive/X-linked fashion. The...

Diseases Caused by Mutations in Mitochondrial Carrier Genes SLC25: A Review.

In the 1980s, after the mitochondrial DNA (mtDNA) had been sequenced, several diseases resulting from mtDNA mutations emerged. Later, numerous disorders caused by mutations in the nuclear genes encoding mitochondrial proteins were found. A group of these diseases are due to defects of mitochondrial carriers, a family of proteins named solute carrier family 25 (SLC25), that transport a variety of solutes such as the reagents of ATP synthase (ATP, ADP, and phosphate), tricarboxylic acid cycle intermediates, cofactors, amino acids, and carnitine esters of fatty acids. The disease-causing mutations disclosed in mitochondrial carriers range from point mutations, which are often localized in the substrate translocation pore of the carrier, to large deletions and insertions. The biochemical consequences of deficient transport are the compartmentalized accumulation of the substrates and dysfunctional mitochondrial and cellular metabolism, which frequently develop into various forms of myopathy, encephalopathy, or neuropathy. Examples of diseases, due to mitochondrial carrier mutations are: combined D-2- and L-2-hydroxyglutaric aciduria, carnitine-acylcarnitine carrier deficiency, hyperornithinemia-hyperammonemia-homocitrillinuria (HHH) syndrome, early infantile epileptic encephalopathy type 3, Amish microcephaly, aspartate/glutamate isoform 1 deficiency, congenital sideroblastic anemia, Fontaine progeroid syndrome, and citrullinemia type II. Here, we review all the mitochondrial carrier-related diseases known until now, focusing on the connections between the molecular basis, altered metabolism, and phenotypes of these inherited disorders.

Novel biallelic TRNT1 mutations lead to atypical SIFD and multiple immune defects

Mutations in the gene encoding transfer RNA (tRNA) nucleotidyltransferase, CCA-adding 1 (TRNT1), an enzyme essential for the synthesis of the 3′-terminal CCA sequence in tRNA molecules, are associated with a rare syndrome of congenital sideroblastic anemia, B cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Clinical manifestations and immunological phenotypes were assessed in a Chinese patient with novel compound heterozygous mutations in TRNT1. The patient required multiple hospitalizations starting at the age of 2 years for recurrent fevers without an infective cause. During the febrile episode, the patient was found to have microcytic hypochromic anemia, B cell lymphopenia, and hypogammaglobulinemia. Targeted gene sequencing identified novel compound heterozygous mutations in the TRNT1 gene (c.525delT, p.Leu176X; c.938T>C, p.Leu313Ser). Immunophenotyping revealed increased CD8+ T cells, CD4+ terminally differentiated effector memory helper T lymphocytes (CD4 TEMRA), and CD4+ effector memory lymphocytes (CD4 EM). Analysis of CD4+ T subsets identified decreased T follicular helper cells (Tfh) with a biased phenotype to Th2-like cells. The patient also showed a lower percentage of switched memory B (smB) cells. Additionally, defects in the cytotoxicity of the patient's NK and γδT cells were shown by CD107alpha expression. In conclusion, TRNT1 mutations may lead to multiple immune abnormality especially humoral and cytotoxicity defects, which indicate that SIFD is not only suffered ‘Predominantly antibody deficiencies’ in IUIS classification system, and further studies are needed to understand the pathogenesis of immunodeficiency in these patients.

Ring sideroblasts and iron metabolism

Ring sideroblasts show abnormal mitochondrial iron accumulation, and their emergence in the bone marrow is a characteristic of sideroblastic anemias (SAs). SAs are a group of heterogeneous congenital and acquired disorders. Congenital SA is a rare disease caused by gene mutations involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur after exposure to certain drugs or alcohol and due to copper deficiency (secondary SA). Furthermore, SAs are associated with myelodysplastic syndrome (idiopathic SA), strongly correlating with specific somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery. Recent reports have indicated that common defects in iron/heme metabolism underlie in the mechanisms of ring sideroblast formation in congenital and acquired SAs. Current understanding of SA pathophysiology, including the mechanisms of ring sideroblast formation, is discussed in this review.

Molecular Characterization and Novel Therapeutic Strategy for X-Linked Sideroblastic Anemia Associated with ALAS2 Missense Variants

Background: Congenital sideroblastic anemia (CSA) is an inherited anemia characterized by the presence of ring sideroblasts (RSs) in bone marrow. X-linked sideroblastic anemia (XLSA) is the most common form of CSA and is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Each ALAS2 mutation has a variable effect on the protein's enzymatic function, and only half of XLSA patients are responsive to pyridoxal 5′-phosphate (PLP) treatment. Thus, novel therapeutic strategies should be explored. We have established an in vitro XLSA model in human induced pluripotent stem cell-derived erythroid progenitors (HiDEP-1) by disrupting GATA-1-binding intronic enhancer region of ALAS2, which accounts for a minor XLSA subtype with PLP refractoriness (Saito and Ono et al. MCB 2019; ASH 2018). However, evidence regarding molecular characterization of ALAS2 missense variants is lacking, presumably due to difficulties in introducing the desired missense mutations in primary erythroblasts. In this study, we optimized a protocol for introducing point mutations in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells and subsequently characterized XLSA models harboring ALAS2 missense variants. Methods: Based on homology-directed CRISPR/Cas9 system, we introduced an ALAS2 mutation from arginine at amino acid residue 170, one of the XLSA hot spots, into leucine (R170L) or histidine (R170H). A twenty-nucleotide targeted genomic sequence, located upstream of protospacer adjacent motif (PAM) near ALAS2 R170, was cloned into plasmid vectors expressing Cas9 nuclease and green fluorescent protein (GFP). Each homologous recombinant template was designed as a 127 single-stranded oligodeoxynucleotide (ssODN) asymmetrically distributed from the Cas9-mediated cleavage site. PAM silent mutations were added to avoid continuous cleavage after successful mutation. HUDEP-2 cells were co-transduced with gRNA/Cas9/GFP-expressing vectors and ssODNs using Amaxa Nucleofector 2b (Lonza). After 48 hours, cells were isolated by GFP-positive expression; subsequently, single-cell dilution was performed, followed by clonal analysis. Results: Both XLSA clones (ALAS2 R170L and R170H) appeared pink/pale colored, reflecting impaired hemoglobin biosynthesis. Consistently, quantitative RT-PCR analysis demonstrated decreased globin gene (HBB) expression. On the other hand, XLSA clones did not show RSs and were morphologically similar to wild-type controls. When XLSA clones were induced to undergo erythroid differentiation by co-culturing with OP9 cells in a 100 mM sodium ferrous citrate (SFC)-supplemented medium, increased numbers of RSs were observed in mutant clones, mimicking findings in XLSA patients. Electron microscopy confirmed an aberrant mitochondrial iron deposit in XLSA clones. Also, HBB expression and intracellular heme concentration were significantly lower than those in wild-type controls. Intriguingly, while OP9 co-culture and SFC addition promoted erythroid differentiation in both wild-type and XLSA clones, XLSA clones exhibited a more immature morphological phenotype than wild-type controls. Expression profiling revealed that 317 and 86 genes were commonly up- and downregulated >2-fold in XLSA clones compared with those in wild-type controls. Gene ontology analysis showed significant (p < 0.01) enrichment of genes associated with mitochondrial gene expression and organization, suggesting that mitochondrial alteration is involved in XLSA pathogenesis. On the other hand, 188 genes in total were differentially expressed between ALAS2 R170L and R170H. Genes associated with ferroptosis, such as GPX4 and GCLC, showed a significantly lower expression in ALAS2 R170H clones, possibly accounting for phenotypic differences among patients with ALAS2 R170L or R170H mutation. Finally, supplementation with 5-aminolevulinic acid (ALA) significantly improved the compromised heme biosynthesis in XLSA clones, suggesting ALA treatment to be a promising therapeutic option for XLSA. Conclusion: The established models may act as useful tools for exploring the precise molecular mechanisms of XLSA harboring missense mutations and for drug testing. Also, our homology-directed CRISPR/Cas9-based protocol can be applied to establish a wide variety of CSA as well as congenital anemia models. Disclosures Fukuhara: Zenyaku: Honoraria; Eisai: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Gilead: Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinkyaku: Honoraria; Celgene Corporation: Honoraria, Research Funding; Mundi: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; AbbVie: Research Funding; Bayer: Research Funding; Solasia Pharma: Research Funding. Onishi:Celgene: Honoraria; Kyowa-Hakko Kirin: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; Nippon Shinyaku: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed >1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p< 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

Hepcidin and Anemia: A Tight Relationship

Hepcidin, the master regulator of systemic iron homeostasis, tightly influences erythrocyte production. High hepcidin levels block intestinal iron absorption and macrophage iron recycling, causing iron restricted erythropoiesis and anemia. Low hepcidin levels favor bone marrow iron supply for hemoglobin synthesis and red blood cells production. Expanded erythropoiesis, as after hemorrhage or erythropoietin treatment, blocks hepcidin through an acute reduction of transferrin saturation and the release of the erythroblast hormone and hepcidin inhibitor erythroferrone. Quantitatively reduced erythropoiesis, limiting iron consumption, increases transferrin saturation and stimulates hepcidin transcription. Deregulation of hepcidin synthesis is associated with anemia in three conditions: iron refractory iron deficiency anemia (IRIDA), the common anemia of acute and chronic inflammatory disorders, and the extremely rare hepcidin-producing adenomas that may develop in the liver of children with an inborn error of glucose metabolism. Inappropriately high levels of hepcidin cause iron-restricted or even iron-deficient erythropoiesis in all these conditions. Patients with IRIDA or anemia of inflammation do not respond to oral iron supplementation and show a delayed or partial response to intravenous iron. In hepcidin-producing adenomas, anemia is reverted by surgery. Other hepcidin-related anemias are the “iron loading anemias” characterized by ineffective erythropoiesis and hepcidin suppression. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and some forms of hemolytic anemias as pyruvate kinase deficiency. The paradigm is non-transfusion-dependent thalassemia where the release of erythroferrone from the expanded pool of immature erythroid cells results in hepcidin suppression and secondary iron overload that in turn worsens ineffective erythropoiesis and anemia. In thalassemia murine models, approaches that induce iron restriction ameliorate both anemia and the iron phenotype. Manipulations of hepcidin might benefit all the above-described anemias. Compounds that antagonize hepcidin or its effect may be useful in inflammation and IRIDA, while hepcidin agonists may improve ineffective erythropoiesis. Correcting ineffective erythropoiesis in animal models ameliorates not only anemia but also iron homeostasis by reducing hepcidin inhibition. Some targeted approaches are now in clinical trials: hopefully they will result in novel treatments for a variety of anemias.

Clinical features and gene mutation spectrum in children with sideroblastic anemia

To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA. Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed. Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic". ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.

Reticulocyte Hemoglobin Equivalent (Ret-He) Combined with Red Blood Cell Distribution Width Has a Differentially Diagnostic Value for Thalassemias

As a type of congenital microcytic hypochromic anemia, thalassemia trait is often confused with other conditions, such as congenital sideroblastic anemia (CSA) and iron deficiency anemia, before a specific work-up is performed. However, these tests, including hemoglobin (Hb) electrophoresis, gene mutations and Prussian blue staining after bone marrow aspirate, are relatively expensive, time-consuming and invasive. To find labor-saving parameters to facilitate differential diagnosis, we retrospectively analyzed the routine blood indexes of 59 thalassemia trait cases [22 α-thalassemia (α-thal), 36 β-thalassemia (β-thal) and one α/β-thal], 21 CSA patients, and 238 iron deficiency anemia controls. Significantly higher reticulocyte Hb equivalent (Ret-He) and lower red blood cell (RBC) distribution width (RDW) were prominent in thalassemia trait. Furthermore, RDW-standard deviation (SD) was independent of the severity of anemia in thalassemia trait, similar to Ret-He in CSA. In the context of the same grades of anemia, Ret-He combined with RDW was powerful in differentiation of thalassemia from CSA and iron deficiency anemia. By receiver operation curve (ROC) analysis, Ret-He had a specificity of 67.06% and a sensitivity of 76.92% with a cutoff value of 20.9 pg for thalassemia trait in mild anemia and a specificity of 84.09% and a sensitivity of 68.42% with a cutoff value of 19.1 pg for thalassemia trait in moderate anemia. Regarding CSA, Ret-He had 92.94% specificity and 60.00% sensitivity in mild anemia, with a cutoff value of 18.1 pg. Overall, Ret-He and RDW are two convenient indexes able to differentiate thalassemia from the other two microcytic anemias, CSA and iron deficiency anemia.

Genotype/phenotype correlations of childhood-onset congenital sideroblastic anaemia in a European cohort.

Congenital sideroblastic anaemia (CSA) is a rare disease caused by germline mutations of genes involved in haem and iron-sulphur cluster formation, and mitochondrial protein biosynthesis. We performed a retrospective multicentre European study of a cohort of childhood-onset CSA patients to explore genotype/phenotype correlations. We studied 23 females and 20 males with symptoms of CSA. Among the patients, the most frequently mutated genes were ALAS2 (n=10; 23·3%) and SLC25A38 (n=8; 18·6%), causing isolated forms of microcytic anaemia of varying severity. Five patients with SLC19A2 mutations suffered from thiamine-responsive megaloblastic anaemia and three exhibited the 'anaemia, deafness and diabetes' triad. Three patients with TRNT1 mutations exhibited severe early onset microcytic anaemia associated with thrombocytosis, and two exhibited B-cell immunodeficiency, inflammatory syndrome and psychomotor delay. The prognoses of patients with TRNT1 and SLC2A38 mutations were generally dismal because of comorbidities or severe iron overload. No molecular diagnosis could be established in 14/43 cases. This study emphasizes the frequency of ALAS2 and SLC25A38 mutations and provides the largest comprehensive analysis to date of genotype/phenotype correlations in CSA. Further studies of CSA patients with data recorded in an international registry would be helpful to improve patient management and establish standardized guidelines.

Porphyries cutanées

The porphyrias are a group of metabolic disorders resulting from an innate abnormality in haem biosynthesis, and the clinical settings of which vary according to the genetic enzyme abnormality in question. These are genetic disorders with autosomal dominant or recessive inheritance of varying penetrance, and whose clinical expression differs according to the preferential location of haem precursors. Different classifications have been proposed according to genetic inheritance, the enzyme anomaly at issue, and clinical expression. The clinical classification distinguishes between acute porphyria (acute intermittent porphyria, porphyria variegata, hereditary coproporphyria), bullous cutaneous porphyrias (porphyria cutanea tarda, porphyria variegata and hereditary coproporphyria), painful photosensitive acute cutaneous porphyrias (erythropoietic protoporphyria and X-linked dominant protoporphyria), and rare recessive porphyrias (congenital erythropoietic porphyria, Doss porphyria, hepatoerythropoietic porphyria and harderoporphyria). Treatment depends on the clinical expression of the disorder.

GLRX5 mutations impair heme biosynthetic enzymes ALA synthase 2 and ferrochelatase in Human congenital sideroblastic anemia

Non-syndromic microcytic congenital sideroblastic anemia (cSA) is predominantly caused by defective genes encoding for either ALAS2, the first enzyme of heme biosynthesis pathway or SLC25A38, the mitochondrial importer of glycine, an ALAS2 substrate. Herein we explored a new case of cSA with two mutations in GLRX5, a gene for which only two patients have been reported so far. The patient was a young female with biallelic compound heterozygous mutations in GLRX5 (p.Cys67Tyr and p.Met128Lys). Three-D structure analysis confirmed the involvement of Cys67 in the coordination of the [2Fe2S] cluster and suggested a potential role of Met128 in partner interactions. The protein-level of ferrochelatase, the terminal-enzyme of heme process, was increased both in patient-derived lymphoblastoid and CD34+ cells, however, its activity was drastically decreased. The activity of ALAS2 was found altered and possibly related to a defect in the biogenesis of its co-substrate, the succinyl-CoA. Thus, the patient exhibits both a very low ferrochelatase activity without any accumulation of porphyrins precursors in contrast to what is reported in erythropoietic protoporphyria with solely impaired ferrochelatase activity. A significant oxidative stress was evidenced by decreased reduced glutathione and aconitase activity, and increased MnSOD protein expression. This oxidative stress depleted and damaged mtDNA, decreased complex I and IV activities and depleted ATP content. Collectively, our study demonstrates the key role of GLRX5 in modulating ALAS2 and ferrochelatase activities and in maintaining mitochondrial function.

Sideroblastic anemia

Sideroblastic anemia (SA) signifies a group of heterogeneous congenital and acquired disorders characterized by anemia and the presence of ring sideroblasts in the bone marrow. Congenital SA is a rare disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. In addition, SA can occur after exposure to certain drugs or alcohol and because of copper deficiency (secondary SA). Of note, SA also correlates with myelodysplastic syndrome (idiopathic SA). Recent progress in the genome analysis technology has enabled the identification of novel causative genes for SA, elucidating the molecular pathophysiology of these disorders. Accordingly, the significance of genetic diagnosis for SA has been increasing. This review discusses the current understanding of genetic mutations involved in the pathophysiology of SA.

Clinical Characterization of a Novel Mutation in SLC25A38 Resulting in Congenital Sideroblastic Anemia in a Canadian First Nations Population

Introduction: Congenital sideroblastic anemia (CSA) is an uncommon cause of inherited anemia characterized by pathologic iron accumulation. Severity of anemia and blood transfusion requirements are variable. Aberrations in iron-heme metabolism that underpin CSA result in iron overload, often preceding transfusion. The only curative therapy for CSA is hematopoietic stem cell transplantation (HSCT), offered to select patients with severe transfusion dependent anemia. Mutations in several genes have been identified to be responsible for CSA. Mutations in SLC25A38, encoding a mitochondrial glycine transporter required for heme biosynthesis, are a rare cause of CSA. Homozygosity for a novel founder missense mutation in SLC25A38 (c.560G>A) has been detected in a Canadian First Nations Northern Cree population. The associated phenotype is a severe anemia presenting in infancy and characterized by ineffective erythropoiesis and iron loading. The natural history and treatment outcomes of CSA caused by the c.560G>A mutation have not been described.Objectives: (1) To characterize the clinical features in patients homozygous for this founder mutation, including propensity to iron loading and (2) to describe the treatment course and outcomes for red cell transfusion, iron chelation, pyridoxine supplementation and HSCT.Methods: A retrospective chart review was conducted at the Winnipeg Health Sciences Centre and CancerCare Manitoba of all affected individuals homozygous for the c.560G>A mutation in SLC25A38.Results: Seven affected individuals (three females and four males) were identified. Comorbidities including dextrocardia, hypertrophic cardiomyopathy, rheumatoid arthritis and global developmental delay were noted in three members of the kindred, but there was no evidence of a syndromic form of CSA. The median age of presentation with anemia was 6 months (24 days - 4.4 years). The median age of CSA diagnosis was 1.5 years (6 months - 22 years). Alternative causes of anemia were initially suspected in five of the seven patients: iron deficiency, resulting in oral iron supplementation, and alpha thalassemia. Uniformly, individuals presented with a microcytic, hypochromic anemia, with elevated ferritin prior to first red cell transfusion. All affected individuals required red cell transfusion support, with six of seven patients requiring chronic transfusions at a median frequency of every four weeks. One individual, though chronically anemic, did not require regular transfusion support until the age of 20 years. One patient carried a successful pregnancy with transfusion support, but delivered prematurely. For all individuals, iron chelation was recommended, with six individuals starting chelation within three years of initiation of transfusions. Hepatic, cardiac, pituitary and pancreatic iron overload, and resultant organ dysfunction were noted in two subjects who did not adhere to chelation; one died of neutropenic sepsis after starting deferiprone for cardiac iron overload. Six individuals were given trials of pyridoxine supplementation, with two demonstrating a transient partial response with a rise in reticulocyte count and decrease in transfusion frequency. Three individuals underwent allogeneic HSCT (two from matched sibling donors, and one from a matched unrelated donor), at 5.5 years, 7.2 years, and 28 years. The oldest and most iron-loaded of the three died in the post-transplant period due to complications of sepsis. The other two individuals remain transfusion free, at 9 months and 15.6 years post-HSCT.Conclusions: A novel founder mutation in SLC25A38 causing CSA among individuals of Canadian First Nations Northern Cree descent results in a severe transfusion-dependent anemia. Despite a common genetic etiology, phenotypic variability was noted, with one individual having marked tolerance to anemia. A partial transient response to pyridoxine was noted in two individuals, raising the question of an alternative role for SLC25A38 in heme biosynthesis. HSCT, when performed before significant iron overloading, was beneficial. Characterization of this phenotype and evidence of successful HSCT may assist clinicians in identifying affected individuals with CSA and initiating timely and effective treatment. DisclosuresNo relevant conflicts of interest to declare.

“in the Patients' Hands”- a Structured Survey of Patient Voices to Prioritise Research Questions in Rare Inherited Anaemias: The James Lind Alliance Priority Setting Partnership

Patients with rare inherited anaemias (RIAs) may take years to receive a precise diagnosis. Lack of equitable access to the few centres of specialist expertise means not all patients will receive the same standard of care. Few treatments have been developed, in part from lack of research. The research agenda is traditionally set by scientists, the medical profession, funding bodies and pharmaceutical companies. The voice of patients and carers is rarely considered in deciding which questions will be addressed in studies. Involving patients in setting the research agenda is likely to improve the validity and quality of research, but the practicalities of surveying patient voices objectively are not straightforward. Using a well-established, structured format for collecting patient-driven priorities, we carried out a James Lind Alliance (JLA) Priority Setting Partnership (PSP) in RIA. This has been used internationally to produce patient-driven research priorities in >60 disorders, eg blood transfusion and donation.The aim of the study was to determine what people with RIAs, their carers and health professionals believe to be the most important areas for future research using a 3-step sequential survey approach. The steering group (SG) comprised adult and paediatric haematologists, nurses, patients, representatives from patient organisations, and a JLA facilitator, reflecting representation from medical and lay stakeholders. The SG chose to focus this piece of work on disorders which are individually rare or very rare to provide the first ever opportunity for a collective voice: Diamond-Blackfan Anaemia, Congenital Dyserythropoietic Anaemia, Congenital Sideroblastic Anaemia, red cell membrane and enzyme disorders, and transfusion-dependent unexplained RIAs. The SG devised the first survey (PSP Survey 1) comprising open questions to address the diagnosis, treatment, and management of these conditions. Survey 1 was distributed UK-wide to adult and paediatric haematologists, patient organisations and support groups and on social media. The British Society of Haematology distributed to all members. Haematologists were invited to send the survey to their patients.88 people responded to PSP Survey 1, listing questions they would most like answered. Of these, 23% were patients, 27% relatives/friends, 44% health/social care professionals, 6% other. A total of 314 valid questions were submitted, then filtered by grouping similar/overlapping questions, removing those answered in the literature, leaving 48 unanswered questions to be taken forward in PSP Survey 2. 120 respondents ranked each question on a Likert scale, indicating the degree of personal importance. Individual scores were totalled to provide a shortlist of 25 questions for Stage 3, a one-day multidisciplinary workshop with 30 people representing the breadth of stakeholders recruited via patient and clinician groups and through social media. The Stage 3 PSP workshop identified the ‘Top 10‘ questions by crossover multidisciplinary mini-working groups reaching a consensus using shared understanding between patients and professionals:Would a national formal network of clinicians with expertise and /or a national multidisciplinary team meeting improve care for patients with (RIA)?Can the diagnostic pathways be improved to provide faster and more accurate diagnoses in a cost effective manner?Could an understanding of the cellular and molecular processes in red blood cell production lead to new treatments?Could the need for iron chelation be reduced? Could current approaches and monitoring be improved?How do existing drugs for RIA work? Could this understanding lead to new treatments and new ways of delivering treatments?How can the fatigue of severe anaemia be managed (apart from blood transfusions)?Would a register of all RIA patients in the UK (including data and samples) improve care?How is quality of life affected by RIA and its treatment? How could this be improved for patients?What factors indicate that a person with a RIA needs a transfusion, and what is the best regime to maintain safety and quality of life?How can high quality care be sustained throughout a patient's lifetime (e.g. from child to adult and into old age)?Engagement by funding bodies to prioritise funding for projects addressing these patient-selected priorities is now key to ensure the success of this initiative. DisclosuresNo relevant conflicts of interest to declare.

Striking Difference in Iron Utilization between Oral and Intravenous Iron in Various Anemias and Hemochromatosis

Introduction Patients with hereditary hemochromatosis (HH) and non-transfusion-dependent hereditary anemia (HA NTD) both express low hepcidin levels, leading to increased intestinal iron absorption and, ultimately, predominantly parenchymal iron overload. Knowledge about iron absorption in humans stems from iron absorption or utilization studies with radio-labeled iron performed in the 60-70s from the last century. Here, we present unique data of combined absorption and utilization studies in a large cohort of patients with primary and secondary hemochromatosis.Methods We retrospectively analyzed the data from iron absorption and kinetics studies performed from 1972 until 1994 as part of routine clinical practice in patients with iron-related health problems at the University Medical Center Utrecht, the Netherlands. A radioactive tracer dose of oral (1 mg) 59Fe with 51Cr as non-absorbable indicator, or intravenous (10 µCi) 59Fe was administered. Radio-activity was measured with a whole-body counter to assess absorption and with a gamma-counter to determine radio-activity in peripheral blood samples to calculate the amount of iron utilized for red blood cell (RBC) production.Main findings Iron absorption was analyzed in 6 distinct groups with and without iron overload and iron reducing therapy (table). Iron uptake is the percentage of the iron test dose taken up by enterocytes, retention the percentage retained in the body 14 days after ingestion, and transfer the fraction of iron taken up that is retained in the body. Iron uptake, transfer and retention were significantly higher in patients with treated and untreated HH and iron deficiency (ID) compared with healthy controls (p<0.01). Notably, uptake, transfer and retention were also significantly higher in the analyses of patients with treated or untreated HA NTD (including 19 congenital sideroblastic anemia, 6 hereditary spherocytosis, 5 congenital dyserythropoietic anemia, 3 non-transfusion dependent thalassemia, 4 Hb Adana, 2 hexokinase deficiency and 1 PKD) than in analyses of healthy controls (p<0.01). Next, iron retention was used to calculate the percentage of iron utilized for RBC production after 14 days. Mean percentages of RBC iron utilization (RBCIU) after an oral iron test dose were 37% (SD 17%) in untreated HA NTD, 53% (SD 19%) in treated HA NTD, 55% (SD 20%) in untreated HH, 70% (SD 22%) in treated HH, and 99% (SD 22%) in ID patients. Surprisingly, RBCIU was lower after oral than after intravenous iron in patients with HA NTD or HH (figure 1). The difference between oral and intravenous RBCIU was expressed as percentage of intravenous RBCIU, and denominated as the LIR (liver iron retention). The LIR had a mean value of 28% (SD 26%) in untreated HH, 23% (SD 24%) in untreated HA NTD, and 16% (SD 25%) in treated HH patients, all significantly higher than the LIR of 1% (SD 22%) measured in ID patients (p<0.05). The LIR was strongly correlated to iron saturation (r=0.41; p<0.01).Main conclusions Of major interest is the observation that a substantial fraction of oral iron retained in patients with iron overload was not utilized for erythropoiesis. Under circumstances of high transferrin saturation, a part of iron transported from enterocytes into the portal circulation will be non-transferrin-bound iron (NTBI). A part of this NTBI will be available as labile plasma iron (LPI), a form of iron with high redox potential, and the capacity to rapidly cross membranes via transporters and channels. Recently it was shown that in iron-overloaded conditions LPI is almost completely taken up after passage of the liver and this is facilitated by ZIP14 in a non-transferrin-dependent way. (Jenkitkasemwong et al. Cell Metabolism, 2015: 22(1), 138-150). We therefore hypothesize that LPI produced primarily in the portal system (oral dosed iron) is primarily taken up in the liver and that LPI produced elsewhere in the circulation (intravenous dosed iron) may be taken up by other organs as well.In conclusion, our data is suggestive of the existence of significant hepatic scavenging of NTBI/LPI under iron-overloaded conditions. This could explain the distinct patterns of transfusion-dependent and transfusion-independent iron overload and we suggest that ZIP14 could facilitate this. [Display omitted] Disclosuresvan Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Molecular analysis of 19 Spanish patients with mixed porphyrias

Porphyrias are rare diseases caused by alterations in the heme biosynthetic pathway. Depending on the afected enzyme, porphyrin precursors or porphyrins are overproduced, causing acute neurovisceral attacks or dermal photosensitivity, respectively. Hereditary Coproporphyria (HCP) and Variegate Porphyria (VP) are mixed porphyrias since they can present acute and/or cutaneous symptoms. These diseases are caused by a deficiency of coproporphyrinogen oxidase (CPOX) in HCP, and protoporphyrinogen oxidase (PPOX) in VP. Herein, we studied nineteen unrelated Spanish patients with mixed porphyrias. The diagnosis of either, HCP or VP was made on the basis of clinical symptoms, biochemical findings and the identification of the mutation responsible in the CPOX or PPOX genes. Two patients presented both acute and cutaneous symptoms. In most patients, the biochemical data allowed the diagnosis. Among eleven patients with HCP, ten CPOX mutations were identified, including six novel ones: two frameshift (c.32delG and c.1102delC), two nonsense (p.Cys239Ter and p.Tyr365Ter), one missense (p.Trp275Arg) and one amino acid deletion (p.Gly336del). Moreover, seven previously described PPOX mutations were identified in eight patients with VP. The impacts of CPOX mutations p.Trp275Arg and p.Gly336del, were evaluated using prediction softwares and their functional consequences were studied in a prokaryotic expression system. Both alterations were predicted as deleterious by in silico analysis. Aditionally, when these alleles were expressed in E. coli, only p.Trp275Arg retained some residual activity. These results emphasize the usefulness of integrated the biochemical tests and molecular studies in the diagnosis. Furthermore, they extend knowledge on the molecular heterogeneity of mixed porphyrias in Spain.

Diverse phenotype in patients with complex I deficiency due to mutations in NDUFB11

Mitochondrial complex I deficiency is the most frequent mitochondrial disorder presenting in childhood and the mutational spectrum is highly heterogeneous. The NDUFB11 gene is one of the recently identified genes, which is located in the short arm of the X-chromosome. Here we report clinical, biochemical, functional and genetic findings of two male patients with lactic acidosis, hypertrophic cardiomyopathy and isolated complex I deficiency due to de novo hemizygous mutations (c.286C > T and c.328C > T) in the NDUFB11 gene. Neither of them had any skin manifestations. The NDUFB11 gene encodes a relatively small integral membrane protein NDUFB11, which is essential for the assembly of an active complex I. The expression levels of this protein was decreased in both patient cells and a lentiviral complementation experiment also supported the notion that the complex I deficiency in those two patients is caused by NDUFB11 genetic defects. Our findings together with a review of the thirteen previously described patients demonstrate a wide spectrum of clinical features associated with NDUFB11-related complex I deficiency. However, histiocytoid cardiomyopathy and/or congenital sideroblastic anemia could be indicative for mutation in the NDUFB11 gene, while the clinical manifestation of the same mutation can be highly variable.

Case Report of Hereditary Coproporphyria Accompanied by Nystagmus and Vestibular Dysfunction

Porphyria is a collection of disorders resulting from a breakdown in heme synthesis. The most common form is acute intermittent porphyria (AIP), and the other disorders are less common and therefore more difficult to diagnose. Our case study focuses on a 39-year-old male with a combination of gastrointestinal symptoms as well as neurocutaneous manifestations. Failing to recognize this disorder in the primary care setting can lead to additional, sometimes repetitive, clinical investigation and negative outcome for patients. The authors believe strongly in the teamwork model for today's medicine. Close communication between specialty and primary care, as well as a broader differential, could have helped achieve an earlier diagnosis and treatment for the patient. J Med Cases. 2018;9(11):355-359 doi: https://doi.org/10.14740/jmc3158w

Porphyrias-what is verified?

Porphyrias are caused by enzyme defects of heme biosynthesis. According to their clinical presentation and to each affected pathway, they are categorized into acute and non-acute as well as hepatic and erythropoietic porphyrias. Acute hepatic porphyrias, e.g. acute intermittent porphyria (AIP), porphyria variegata (VP), hereditary coproporphyria (HCP) and 5‑aminolevulinic acid dehydratase-deficient porphyria (ALADP) are characterized by accumulation of the porphyrin precursors 5‑aminolevulinic acid (ALA) and porphobilinogen (PBG) that correlate with severe abdominal, psychiatric, neurological or cardiovascular symptoms. Additionally, skin photosensitivity can occur in VP and less frequently, in HCP. Decisive for the diagnosis of acute hepatic porphyrias are a >4-fold elevated urinary excretion of ALA in ALADP and ALA and PBG in all other acute porphyrias. First-line treatment of an acute porphyria attack includes intensive care with pain management, sufficient caloric supply, strict avoidance of porphyrinogenic drugs and elimination of other triggering factors. Heme therapy is indispensable in case of developing neurological symptoms and clinical worsening despite first-line measures. Non-acute porphyrias, mainly porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP) and X‑linked protoporphyria (XLP) display accumulation of porphyrins in the skin and/or liver resulting in photosensitivity up to possible liver damage. Patients with PCT benefit from iron depletion, low-dose chloroquine treatment and/or hepatitisC virus elimination. Afamelanotide is associated with better sunlight tolerance in patients with EPP and XLP. Moreover, innovative therapies that highly selectively address dysregulated steps of the heme biosynthetic pathway are currently under clinical trial.